Using a concurrent challenge with porcine circovirus 2 and porcine reproductive and respiratory syndrome virus to compare swine vaccination programs

The objectives of the present study were to evaluate the immune response of six commercial vaccines against PRRSV-2 and PCV2, administered as monovalent or combined products via intramuscular (IM) or intradermal (ID) routes. Seventy-two, 3-week-old pigs were randomly allocated into 8 treatments with 9 pigs each: IMPP0/PCVMH7, IDPP0/PCVMH7, IMING0/PCVMH7, IMPP0/PCVMH0, IDPP0/PCVMH0, IMTRF0, NV/CH, and NV/NC. IMPP0/PCVMH0 and IMPP0/PCVMH7 groups were IM vaccinated once with Prime Pac PRRS (MSD Animal Health, The Netherlands) at 0 days post-vaccination (DPV), followed by single IM vaccination with Porcilis PCV M Hyo (MSD Animal Health, The Netherlands) either at 0 or 7 DPV, respectively. IDPP0/PCVMH0 and IDPP0/PCVMH7 groups were ID vaccinated once with Prime Pac PRRS (MSD Animal Health, The Netherlands) at 0 DPV, followed by a single concurrent ID injection of Porcilis PCV ID (MSD Animal Health, The Netherlands) and Porcilis M Hyo ID ONCE (MSD Animal Health, The Netherlands) either at 0 or 7 DPV, respectively. The IMING0/PCVMH7 group was IM vaccinated once with Ingelvac PRRS MLV (Boehringer Ingelheim, Germany) at 0 DPV, and subsequently IM vaccinated with Ingelvac CircoFLEX (Boehringer Ingelheim, Germany) and Ingelvac MycoFLEX (Boehringer Ingelheim, Germany) at 7 DPV. The IMTRF0 group was IM vaccinated once with combined products of Ingelvac PRRS MLV (Boehringer Ingelheim, Germany), Ingelvac CircoFLEX (Boehringer Ingelheim, Germany), and Ingelvac MycoFLEX (Boehringer Ingelheim, Germany) at 0 DPV. The NV/CH and NV/NC groups were left unvaccinated. At 28 DPV (0 days post-challenge, DPC), pigs were intranasally inoculated with a 4 ml of mixed cell culture inoculum containing HP-PRRSV-2 (105.6 TCID50/ml) and PCV2d (105.0 TCID50/ml). Antibody response, IFN-γ-secreting cells (SC), and IL-10 secretion in supernatants of stimulated PBMC were monitored. Sera were collected and quantified for the PRRSV RNA and PCV2 DNA using qPCR. Three pigs from each group were necropsied at 7 DPC, lung lesions were evaluated. Tissues were collected and performed immunohistochemistry (IHC). Our study demonstrated that concurrent vaccination via the ID or the IM route did not introduce additional reactogenicity. We found no interference with the induction of immune response between vaccination timing. In terms of an immune response, ID vaccination resulted in significantly lower IL-10 levels and higher IFN-γ-SC values compared to the IM-vaccinated groups. In terms of clinical outcomes, only one IM-vaccinated group showed significantly better efficacy when antigens were injected separately compared with concurrently. While the vaccines were ID delivered, these effects disappeared. Our findings confirm that concurrent vaccination of PRRSV-2 MLV and PCV2 via either the IM or the ID routes could be a viable immunization strategy to assist with the control of PRDC. In situations where maximal efficacy is required, over all other factors, concurrent vaccination is possible with the ID route but might not be an ideal strategy if using the IM route.

www.nature.com/scientificreports/ immune response post-vaccination with multiple antigens. Therefore, the rationale for this study is to evaluate the immune response of experimental pigs in a model mimicking field conditions with different vaccination timing and routes against PRRSV-2 and PCV2, followed by evaluating the protection level of these strategies with a simultaneous PRRSV-2/PCV2 challenge.

Materials and methods
Experimental design. All animal procedures in the present study were conducted in accordance with the recommendation in the Guild for the Care and Use of Laboratory Animal of the National Research Council of Thailand according to protocols reviewed and approved by the Chulalongkorn University Animal Care and Use Committee (protocol number 2031015). All methods were performed in accordance with the relevant guidelines and regulations. The study is reported in accordance with the ARRIVE guidelines (https:// arriv eguid elines. org). Seventy-two, castrated male, PRRSV-free with 3-week-old pigs were purchased from PRRSV-free commercial herds. Upon arrival, pigs were acclimated for 3 days and tested for the presence of PRRSV and other pathogens with real-time PCR and ELISA kits. Pigs were randomly allocated into 8 treatment groups with 9 pigs each: IMPP0/PCVMH7, IDPP0/PCVMH7, IMING0/PCVMH7, IMPP0/PCVMH0, IDPP0/PCVMH0, IMTRF0, NV/ CH, and NV/NC (Table 1).
Pigs in the IMPP0/PCVMH0 and IMPP0/PCVMH7 groups were intramuscularly (IM) vaccinated once with a 1 ml dose of Prime Pac PRRS (PRRSV-2 MLV, MSD Animal Health, Boxmeer, The Netherlands) at 0 days postvaccination (DPV), followed by a single IM vaccination once with a 2 ml dose of Porcilis PCV M Hyo (MSD Animal Health, Boxmeer, The Netherlands), either at 0 or at 7 DPV, respectively. In contrast, pigs in the IDPP0/ PCVMH0 and IDPP0/PCVMH7 groups were intradermally (ID) vaccinated once with a 0.2 ml dose of Prime Pac PRRS (PRRSV-2 MLV, MSD Animal Health, Boxmeer, The Netherlands) at 0 DPV, followed by concurrent ID vaccination once with a 0.2 ml dose of Porcilis PCV ID (MSD Animal Health, Boxmeer, The Netherlands) and a 0.2 ml dose of Porcilis M Hyo ID ONCE (MSD Animal Health, Boxmeer, The Netherlands), either at 0 or at 7 DPV, respectively. ID vaccination was performed using IDAL 3G needle-free device (MSD Animal Health, Boxmeer, The Netherlands).
At 28 DPV (0 days post-challenge, DPC), pigs were intranasally inoculated with a 4 ml of mixed cell culture inoculum containing HP-PRRSV-2 (FDT10US23 isolate, fifth passage of MARC-145 cells at 10 5.6 TCID 50 /ml) and PCV2 (S1918STC isolate, third passage of PK-15 cells at 10 5.0 TCID 50 /ml), with 2 ml/nostril. Pigs in the NV/NC group were left as a non-vaccination, non-challenge control group. All pigs were housed in separated rooms with separated airflow spaces. Clinical signs and rectal temperatures were daily recorded throughout the experiment.
Blood samples were collected at 0, 7, 14, 21 and 28 DPV, 3, 5, 7, 10, 14, 21 and 28 DPC. Sera were separated and assayed for the presence of PRRSV-and PCV2-specific antibodies using ELISA kits. Genomic copy numbers of PRRSV and PCV2 were quantified using RT-qPCR. Peripheral blood mononuclear cells (PBMC) were isolated and used for in vitro stimulation to measure IL-10 secretion using an ELISA kit and virus-specific IFNγ-secreting cells (IFN-γ-SC) using an ELISPOT assay. At 7 DPC, three pigs from each group were euthanized and necropsied. Pneumonic lung lesions were evaluated as previously described 44 . Tissues were collected, PRRSV-and PCV2-specific antigens were evaluated using immunohistochemistry (IHC). Table 1. Experimental design in the present study. Eight treatment groups include 6 vaccinated groups and 2 non-vaccinated groups. Routes of vaccine administration were included either intramuscular (IM) or intradermal (ID) using IDAL 3G vaccinator. DPV days post-vaccination. The combined vaccine was prepared as follow: Ingelvac MycoFLEX (batch number 2730552A) and Ingelvac CircoFLEX (batch number 3091225A) were mixed, and the mixture was then used to rehydrate Ingelvac PRRS MLV (batch number 2451281A). This was done in place of the Ingelvac PRRS MLV accompanying vaccine diluent and against the manufacturer's mixing condition. A 2 ml dose of combined vaccine was used for IM vaccination.
Homologous vaccine viruses, highly pathogenic (HP)-PRRSV-2 and PCV2 isolates were used in the present study. Homologous vaccine viruses refer to vaccine strains that were used as recall antigens for in vitro stimulation assay in the measurement of virus-specific IFN-γ-SC and IL-10 secretion were performed in previously described methods 24,25 . To challenge pigs, a mixed cell culture supernatant containing Thai HP-PRRSV-2 isolate FDT10US23 (fifth passage in MARC-145 cells) and PCV2 isolate S1918STC (third passage in PK-15 cells) was used as a virus inoculum. The isolate FDT10US23 is an HP-PRRSV-2 variant genetically classified in the sublineage 8.7/HP-PRRSV-2 based on international systematic classification, according to the previously described method 45 . The ORF5 genome sequence is available in GenBank under accession number JN255836. The isolates FDT10US23 and S1918STC were isolated from swine herds experiencing PRRS outbreaks in the western region of Thailand during 2010-2011 46,47 . Pathogenesis and challenge studies of the challenged isolate were demonstrated according to previous studies 25,30,46,48,49 . Clinical signs and rectal temperature recording. Clinical sings and rectal temperature were monitored daily post-vaccination (DPV) and post-challenge periods (DPC) following two consecutive weeks by the same personnel at the same time. The severity of respiratory disease was evaluated using a scoring system for each pig following stress induction as previously described 44 : 0 = normal, 1 = mild dyspnea and/or tachypnea when stressed, 2 = mild dyspnea and/or tachypnea when at rest, 3 = moderate dyspnea and/or tachypnea when stressed, 4 = moderate dyspnea and/or tachypnea when at rest, 5 = severe dyspnea and/or tachypnea when stressed, and 6 = severe dyspnea and/or tachypnea when at rest. Antibody detection. PRRSV-and PCV2-specific antibodies were measured using commercially available ELISA kits: IDEXX PRRS X3 Ab test (IDEXX Laboratories Inc., MA, USA) and BioCheck PCV2 ELISA (Bio-Check BV, Reeuwijk, The Netherlands), and serum neutralization (SN) assay. The ELISA assays were performed following the manufacturer's recommendations. Sera were considered positive for PRRSV if the S/P ratio was greater than 0.4 and positive for PCV2 if the titer was greater than 1070, respectively.

Isolation of porcine peripheral blood mononuclear cells (PBMC). Peripheral blood mononuclear
cells (PBMC) were isolated from heparinized blood using gradient density centrifugation (Lymphosep™, Biowest, MO, USA) as previously described 39 . Isolated PBMC were counted by an inverted microscope, and cell concentrations were accessed in cRPMI-1640 medium [RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, and 50 μg/ml of gentamycin]. The viability of isolated PBMC was determined by Trypan blue (Sigma-Aldrich, MO, USA) staining and more than 90% viability was used for in vitro stimulation for IL-10 production and enzyme-linked immunospot (ELISPOT) assay as described below.
Porcine interleukin-10 (IL-10). Porcine IL-10 concentration in the supernatant of stimulated PBMC was quantified using a porcine ELISA IL-10 kit (R&D System, MN, USA) under the manufacturer's instructions and previously described methods 43,48 . In brief, total 2 × 10 6 PBMC were seed into 96-well plates and cultured in vitro for 24 h with either homologous vaccine viruses at 0.01 multiplicity of infection (MOI), phytohemagglutinin (PHA, 10 μg/ml, Sigma-Aldrich, MO, USA), or MARC-145 cell lysate (mock suspension). In each pig, the levels of porcine IL-10 secretion were calculated by subtracting the value of mock-stimulated well from the PRRSVstimulated well. Subtracted values were compared between treatment groups. ELISPOT assay. The number of virus-specific IFN-γ-SC were determined in stimulated PBMC using a commercial ELISPOT IFN-γ kit (ELISpot porcine IFN-γ, R&D System, MN, USA). The assay was performed in accordance with manufacturer's instructions and a previously described method 43 www.nature.com/scientificreports/ were seeded into 96-well plates and stimulated with either homologous vaccine viruses or heterologous viruses (FDT10US23 and S1918STC isolates) at 0.01 MOI for 24 h. Phytohemagglutinin (PHA) and cRPMI-1640 were used as positive and negative controls, respectively. The spots were counted by an automated ELISPOT Reader (AID ELISPOT Reader, AID GmbH, Strassberg, Germany). The background values were subtracted from the respective count of the stimulated cells and the immune response was expressed as the number of virus-specific IFN-γ-SC per 10 6 PBMC.
Quantification of PRRSV RNA. Total RNA was extracted from sera using NucleoSpin RNA Virus (Macherey-Nagel, Duren, Germany) according to the manufacturer's instructions. The RNA quality was measured using a NanoDrop spectrophotometer (Colibri spectrophotometer, Titertek Berthold, Pforzheim, Germany). Copy number of PRRSV RNA in sera and nasal swabs was quantified using probed-based real-time PCR as previously described 48  Pathological examination. Pigs from each group were necropsied at 7 DPC. Macroscopic and microscopic lung lesions associated with PRRSV-induced pneumonia were evaluated as previously described 44 . For macroscopic lung lesions, each lung lobe was assigned a number to represent the approximate percentage of the volume of the entire lung and the percentage of the volume from each lobe added to the entire lung score (range from 0 to 100% of affected lung). Additionally, the size of lymph nodes was scored ranging from 0 (normal) to 3 (four times the normal size) according to previously described method 50 . Sections were collected from all lung lobes and lymph nodes as previously described 44,50 and fixed with 10% neutral buffered formalin for 7 days. Tissues were routinely processed and embedded in paraffin in an automated tissue processor. Sections were cut and stained with hematoxylin and eosin (H&E). For microscopic lung lesions, the lung sections were examined in a blinded manner and given an estimated score of the severity of interstitial pneumonia. In brief, 0 = normal; 1 = mild interstitial pneumonia; 2 = moderate multifocal interstitial pneumonia; 3 = moderate diffuse interstitial pneumonia, and 4 = severe diffuse interstitial pneumonia. The mean values of the microscopic lung lesion score of each group were calculated.

Immunohistochemistry (IHC).
Immunohistochemistry for PRRSV antigens in lung sections were performed using PRRSV-specific monoclonal antibody as previously described 25 . In brief, tissues were processed and placed on Superfrost Plus slides (Thermo Fisher Scientific, MA, USA). Sections were deparaffinized, rehydrated, and treated with proteinase K (Thermo Fisher Scientific, MA, USA). Endogenous alkaline phosphatase was quenched with 0.3% hydrogen peroxide. Slides were treated with BSA and incubated with monoclonal antibody overnight. After washing, PRRSV antigens were visualized by secondary antibody HRP-conjugated (Agilent, Santa Clara, CA, USA) and counterstained with Meyer's hematoxylin. To obtain quantitative data, slides were analyzed with the NIH Image J 1.50i Program (https:// rsb. info. nih. gov/ ij). In each slide, 10 fields were randomly selected, and the number of positive cells per unit area (0.95 mm 2 ) was determined as previously described 25,51,52 . The mean values were calculated. PCV2 antigens were analyzed by IHC using paraffin-embedded blocks of tissues including lungs, tonsils, tracheobronchial, and inguinal lymph nodes. In brief, tissues were processed and placed on Superfrost Plus slides (Thermo Fisher Scientific, MA, USA). Sections were deparaffinized, rehydrated, and treated with proteinase K (Thermo Fisher Scientific, MA, USA). Endogenous alkaline phosphatase was quenched with 0.3% hydrogen peroxide. Slides were treated with BSA and incubated with PCV-2 specific monoclonal antibody (PCV-2-A, RIT, SD, USA) overnight. After washing, PCV2 antigens were visualized by secondary antibody HRP-conjugated (Agilent, Santa Clara, CA, USA) and counterstained with Meyer's hematoxylin.
The scoring of PCV2 antigen was performed according to previously described 50 . Assessment of staining for PCV2 antigen was done in a blinded fashion and scores ranged from 0 to 3 (0 = negative; 1 = less than 10% of the lymphoid follicles have cells with PCV2 antigen staining; 2 = 10-50% of the lymphoid follicles contain cells with PCV2 antigen staining; 3 = more than 50% of the lymphoid follicles contain cells with PCV2 antigen. Mean values were calculated. Statistical analysis. Analysis of variance (ANOVA) was performed to determine if there were significant differences among groups for each day separately. If the p-value for an ANOVA table was less than or equal to 0.05, the difference between treatment groups was evaluated using a multiple comparison test. All data were analyzed using IBM SPSS Statistic for Windows version 22.0 (IBM, Armonk, NY, USA) (https:// www. ibm. com/ softw are/ analy tics/ spss/ regis ter/).

Results
Rectal temperature and clinical sings. No clinical signs were observed in the NV/NC group throughout the experiment. Following vaccination, the rectal temperature of pigs in all vaccinated groups were in a physiological range and showed no clinical signs. Following challenge, significant differences in the rectal temperature among groups were observed from 4 to 7 DPC, and ID-vaccinated groups had significantly (p < 0.05) lower rectal temperatures compared to that of the IM-vaccinated groups from 6 to 7 DPC. The IMING0/PCVMH7 and NV/ CH groups showed mild-to severe respiratory disease and had significantly (p < 0.05) higher rectal temperatures than that of the other groups at 4 and 5 DPC (Fig. 1). At 6 and 7 DPC, pigs in the IDPP0/PCVMH7 and IDPP0/ PCVMH0 groups had significantly (p < 0.05) lower rectal temperatures compared to not only the IM-vaccinated groups, but also compared with that of the NV/NC group. There was no difference in the rectal temperature among groups from 8 to 28 DPC.
PRRSV-specific antibody response. PRRSV-specific antibody response as measured by ELISA was shown in Fig. 2A. Regardless of vaccine use, route, and timing of administration, all vaccinated groups had slightly increased the PRRSV-specific antibody response at 7 DPV but remained negative for PRRS (S/P ratio < 0.4). Antibody titers continually increased at 14 DPV and reached high levels until 28 DPC. Following vaccination, there was no difference in the antibody response among vaccinated groups from 7 to 28 DPV. Except for the IMPP0/PCVMH7 group, the antibody response was significantly (p < 0.05) lower than that of other groups at 14 and 21 DPV. Following challenge, the PRRSV-specific antibody response was not different among vaccination groups at 7 DPC. At 14 DPC, the IDPP0/PCVMH7, IMING0/PCVMH7, and IDPP0/PCVMH0 groups had significantly (p < 0.05) higher antibody titers than that of the other vaccinated groups. Meanwhile, the IMTRF0 group had a significantly (p < 0.05) lower antibody titer than that of the other vaccinated groups at 14 DPC. There was no difference in the antibody response among vaccinated groups at 21 and 28 DPC. Except for the IMTRF0 group, the antibody response was significantly (p < 0.05) lower than that of the other vaccinated groups at 21 and 28 DPC ( Fig. 2A). No antibody titers were detected in pigs of the NV/NC group throughout the experiment.
PCV2-specific antibody response. PCV2-specific antibody response as measured by ELISA was shown in Fig. 2B. PCV2-specific antibodies were detected in all vaccinated groups at 0 DPV in which the IMPP0/ PCVMH7, NV/CH, and NV/NC groups had significantly (p < 0.05) higher titers than that the IDPP0/PCVMH7, IMING0/PCVMH7, IMPP0/PCVMH0, and IMTRF0 groups. The PCV2-specific antibody titers of the NV/CH and NV/NC groups continually decreased to basal levels over time until 28 DPC. Following vaccination, similar patterns of PCV2 antibody response were detected among vaccinated groups which continually decreased from 7 DPV and dropped to the basal levels at 28 DPV regardless of the vaccine use, route, and timing of administration. Except for one ID-vaccinated group, the slightly increased PCV2 antibody was detected in the IDPP0/ PCVMH0 group at 7 DPV. The PCV2-specific antibody titers of the IDPP0/PCVMH7, IMING0/PCVMH7, IMPP0/PCVMH0, and IMTRF0 groups were significantly (p < 0.05) lower than that of the other groups at 7 DPV. At 14 DPV, the IDPP0/PCVMH7, IMING0/PCVMH7, and IMTRF0 groups had significantly (p < 0.05) lower PCV2-specific antibody titers than that the other groups. Whereas the IDPP0/PCVMH7 and IMTRF0 groups had significantly (p < 0.05) lower PCV2 antibody titers than that of the other groups at 21 and 28 DPV. Following challenge, increased PCV2-specific antibody titers were notably observed in all vaccinated groups. Except for the IMING0/PCVMH7 group that showed decrease in the PCV2 antibody titer until 28 DPC. At 7 DPC, the IMTRF0 group had a significantly (p < 0.05) lower PCV2 antibody titer than that of the other groups. www.nature.com/scientificreports/ Pigs in the IMPP0/PCVMH7, IMPP0/PCVMH0, and IDPP0/PCVMH0 groups had significantly (p < 0.05) higher PCV2 antibody titers than that of the other groups from 14 to 21 DPC. While the PCV2 antibody titers of IMING0/PCVMH7 and IMTRF0 groups had significantly (p < 0.05) lower levels compared to that of the other groups at 28 DPC (Fig. 2B).
Porcine IL-10 production. Increased IL-10 levels were first detected in all vaccinated groups at 7 DPV and continually decreased to basal levels until 28 DPV (Fig. 3). The induction of IL-10 secretion from the cell culture supernatants of stimulated PBMC was markedly detected in the IM-vaccinated groups that reached the highest values at 7 DPV then gradually declined until 28 DPV. Meanwhile, IL-10 secretion in the ID-vaccinated groups continually increased and reached peaks at 14 DPV. The IL-10 levels then continually decreased to the basal levels at 28 DPV. Notably, concurrent vaccination within the same day (day 0/0) showed relatively higher IL-10 secretion than that of the vaccination at day 0/7. At 7 DPV, pigs in the IMTRF0 group had the highest (p < 0.05) IL-10 levels (47.3 ± 4.19 pg/ml) compared to those of the other groups. Meanwhile, the IDPP0/PCVMH7 group had the significantly lowest IL-10 level (9.3 ± 1.6 pg/ml) than that of the other vaccinated groups at 7 DPV. Pigs in the IMING0/PCVMH7 and IMPP0/ PCVMH0 groups had significantly higher IL-10 levels (40.2 ± 2.4 and 37.3 ± 4.0 pg/ml) compared to that of  34.3 ± 5.9 pg/ml) than that of the other groups. At 21 DPV, the IMPP0/PCVMH7 group was the lowest in IL-10 levels (5.3 ± 1.0 pg/ml) than that of the other groups. While the IL-10 secretion in the IMPP0/PCVMH7, IMPP0/ PCVMH0, IDPP0/PCVMH7 and IDPP0/PCVMH0 groups were significantly (p < 0.05) lower levels than that of the other groups at 28 DPV (Fig. 3).

PRRSV-specific IFN-γ-SC.
No PRRSV-specific IFN-γ-SC detected in the NV/CH and NV/NC groups throughout the experiment. The induction of PRRSV-specific IFN-γ-SC was remarkedly observed in the IDvaccinated groups regardless of vaccine use and timing of administration that showed a significantly higher frequency compared to that of the IM-vaccinated group (Fig. 4). For homologous vaccine virus stimulation (Fig. 4A), the PRRSV-specific IFN-γ-SC were first detected at 21 DPV in all vaccinated groups, but the levels were low (ranged from 16.7 ± 14.5 to 36.7 ± 16.7 cells/10 6 PBMC). Then, the frequencies of PRRSV-specific IFN-γ-SC were gradually increased from 21 DPV until 7 DPC in which statistical differences were observed at 28 (Fig. 4A).

PCV2-specific IFN-γ-SC.
No PCV2-specific IFN-γ-SC against heterologous PCV2 recall antigen detected in the NV/CH and NV/NC groups throughout the experiment. The IFN-γ-SC against heterologous PCV2 recall antigen (S1918STC) were relatively low levels compared with the PRRSV recall antigens (Fig. 4C). PCV2-specific IFN-γ-SC were first detected at 28 DPV and reached the peaks at 7 DPC. Pigs in the IDPP0/PCVMH0 and IMTRF0 groups had significantly (p < 0.05) higher frequencies of PCV2-specific IFN-γ-SC (76.67 ± 27.2 and 103.3 ± 42.5 cells/10 6 PBMC) than that of the other groups at 28 DPV. In contrast, the IDPP0/PCVMH7 group had a significantly (p < 0.05) higher frequencies of PCV2-specific IFN-γ-SC (166.7 ± 51.7 cells/10 6 PBMC) than that of the other groups at 7 DPC.  www.nature.com/scientificreports/ cantly (p < 0.05) higher PRRSV RNA at 364.1 ± 97.2 copies/ml than those in the other vaccinated groups. At 14 DPV, the IMPP0/PCVMH0 group had a significantly (p < 0.05) lower PRRSV RNA level (68.55 ± 13.6 copies/ml) compared to those in the other vaccinated groups. There was no statistical difference in the PRRSV RNA levels between groups from 21 to 28 DPV. Following challenge, the PRRSV RNA in the blood rapidly increased and reached peaks at 5 and 7 DPC. Then, the PRRSV RNA levels rapidly decreased to the basal levels from 10 to 28 DPC. There was no difference in PRRSV RNA levels among challenged groups at 3 DPC but an increase in PRRSV RNA levels was detected in all challenged groups at 5 DPC. At 5 DPC, the IMPP0/PCVMH7 group had a significantly (p < 0.05) lower PRRSV RNA (67.8 ± 18.3 copies/ml) than those in the other challenged groups. The reduction of PRRSV RNA in the blood was markedly observed among vaccinated groups from 7 to 10 DPC. At 7 DPC, the IMING0/PCVMH7 and IMTRF0 groups had significantly (p < 0.05) higher PRRSV RNA values (871.1 ± 118.2 and 831.2 ± 128.8 copies/ ml) than that of the other vaccinated groups. In contrast, pigs in the IMPP0/PCVMH7 and IDPP0/PCVMH7 groups (154.6 ± 83.1 and 147.1 ± 30.3 copies/ml) had significantly (p < 0.05) lower PRRSV RNA values than those in the other groups at 7 DPC. At 10 DPC, pigs in the IMING0/PCVMH7 and IMTRF0 groups had significantly  .9 ± 12.9 copies/ml) than that of the other challenged groups. There was no statistical difference in the PCV2 DNA among groups from 21 to 28 DPC (Fig. 5B).

Macroscopic and microscopic lung lesions.
Macroscopic and microscopic lung lesions are summarized in Table 2. The macroscopic lung lesions induced by PRRSV were characterized by multifocal, tan-molted areas with irregular and indistinct borders. The reduction of lung lesions was observed in all vaccinated groups regardless of the vaccine use and time of administration, except for the IMING0/PCVMH7 and IMTRF0 groups. At 7 DPC, pigs in the IMING0/PCVMH7 and IMTRF0 groups showed no difference in macroscopic lung lesion scores compared with the NV/CH group and had significantly (p < 0.05) higher macroscopic lung lesion scores of 63.0 ± 12.5 and 65.0 ± 13.5 than those in the other vaccinated-, challenged groups. In addition, pigs in the IMPP0/PCVMH7 group had a significantly (p < 0.05) lower macroscopic lung lesion score (23.6 ± 13.5) than that of the other challenged groups at 7 DPV. For microscopic lung lesion scores, the lesions were characterized by thickened septa with an increased number of interstitial macrophages and lymphocytes and by type II pneumocyte hyperplasia. Like macroscopic lung lesion scores, pigs in the IMING0/PCVMH7 and IMTF0 groups (1.63 ± 0.22 and 2.33 ± 0.12) had significantly (p < 0.05) microscopic lung lesion scores than in the IDPP0/PCVMH7 and IDPP0/PCVMH0 groups (1.33 ± 0.16 and 1.36 ± 0.17). While the microscopic lung lesion score of pigs in the IMPP0/PCVMH7 group was the lowest (1.15 ± 0.16) compared to that of the other groups (Table 2). Immunohistochemistry (IHC). The mean value of PRRSV-and PCV2-specific antigen scores in tissues are summarized in Table 2. Pigs in the NV/CH group had significantly (p < 0.05) higher PRRSV-and PCV2 antigen scores (15.6 ± 1.6 and 3.30 ± 0.2) than those in the other challenged groups. In accordance with the macroscopic and microscopic lung lesion scores, the Prime Pac vaccination groups showed significantly lower antigen scores than that of the Ingelvac vaccinated groups regardless of the vaccine use and time of administration. For PRRSV antigen score, pigs in the IMPP0/PCVMH7, IDPP0/PCVMH7, IMPP0/PCVMH0 and IDPP0/PCVMH0 groups had significantly (p < 0.05) lower antigen scores of 3.6 ± 0.5, 4.3 ± 0.6, 4.3 ± 0.1 and 5.2 ± 0.3 than that in the IMING0/PCVMH7 and IMTRF0 groups (9.2 ± 0.3 and 9.8 ± 0.5) at 7 DPC. In addition to PRRSV antigen score, pigs in the IMING0/PCVMH7, IDPP0/PCVMH7, IMING0/PCVMH0 and IDPP0/PCVMH0 groups had significantly (p < 0.05) lower PCV2 antigen scores of 0.82 ± 0.06, 0.93 ± 0.16 and 1.05 ± 0.42 and 1.06 ± 0.17 than that in the IMING0/PCVMH7 (1.33 ± 0.32), and IMTRF0 groups (1.53 ± 0.32).

Discussion
Analogous to the vaccination of juvenile swine at weaning, vaccination is commonly practiced in pediatric immunization programs around the world. Children may be vaccinated with different antigens in different limbs simultaneously and this is known as concurrent vaccination. Concurrent vaccination with different antigens has been demonstrated to lead to suboptimal immune responses in some pediatric settings [34][35][36] . This has not been www.nature.com/scientificreports/ reliably demonstrated in swine vaccination and it is highly likely that such interactions are likely to be vaccine component dependent. Therefore, it is critical to evaluate all possible combinations individually, ideally in field studies to understand the immune responses. The present study investigated the immune response of concurrent vaccination with different modified-live PRRSV-2 vaccines, PCV2a-based, and Mycoplasma (M.) hyopneumoneae subunit vaccines when administrated via intramuscular or intradermal routes. In addition to the immune response, the protective efficacy against co-challenge with HP-PRRSV-2 and PCV2 was also evaluated.
Increasing anti-PRRSV antibody responses detected by ELISA from 7 to 14 days post-vaccination are a common feature of PRRSV MLV immunization 24,48 , and in most groups, challenge had a boosting effect on their production. Administration route does not seem to influence this response. Even though ELISA-detected antibodies are not correlated with PRRSV protection (contrarily to viral neutralizing antibodies), it is remarkable that challenge did not result in a boosted antibody response in the IMTRF0 group, which is the one with the most severe lung lesion scores at 7 days post-challenge. In this group, ELISA-detected anti-PRRSV antibody titers were significantly lower than in the other vaccinated groups. This might point to a limited ability of the corresponding vaccine at triggering an appropriate Th2 response in pigs. Sero-neutralizing titers are not significantly lower in this group (data not shown), which further documents that Th2 response measured by ELISA is not a correlate to protection.
All piglets included in the study presented detectable anti-PCV2 antibodies on the day of vaccination. The kinetics observed in the NV/NC group are concordant with the presence of maternally derived antibodies (MDA) decaying over the next 4 weeks. Conventional piglets were used in the present trials and nearly all sow farms in Thailand use quarterly mass-vaccination protocols. Thus, the presence of MDA at the start of the study was to be expected. A significant increase in ELISA antibodies titers was observed on 7 DPV in one vaccinated group, being administered on the same single day: IDPP0/PCVMH0. This increase was transient, but the boosting effect of PCV2 challenge was also significant in this group. In two other vaccinated groups (IDPP0/PCVMH7 and IMTRF0), the boosting effect of PCV2 challenge was more limited and delayed to weeks post-inoculation, without obvious association with PCV2 IHC scores. These two groups were those with the lowest MDA levels at challenge, but whether this is an incidental observation or has a causal link with antibody response would need further exploration.
In all vaccination groups, immunization was followed by a limited viraemia, as is classically described for PRRSV MLV vaccines 53,54 . Post immunization viremia levels were broadly similar across vaccines and administration routes across the 4-week post vaccination period. Simultaneous challenge with an HP-PRRSV-2 strain and PCV2 of groups of pigs with different vaccination protocols/administration routes shows that all groups (except the non-vaccinated) develop comparable viraemia profiles, as well as specific antibody responses. However, in most cases, the ID administration route, irrespective of the vaccine brand, provides improved post-challenge growth performance and significantly decreased lung histopathological scores. Also, ID vaccination seems to induce an earlier development and a significantly higher level of activation of the IFN-γ-SC, both against homologous and heterologous HP-PRRSV-2 viruses. The IMPP0/PCVMH0 group showed a relatively lower ADWG over the 4 weeks post-vaccination period compared to other groups (data not shown). However, this was not reflected in the immune responses or the viremia levels of either PCV2 or PRRSV, suggesting that other factors may have contributed to the slower growth rate in this group. This confirms that all commercial vaccines are well-tolerated by the pigs and have no deleterious impact on growth, irrespective of their administration route. While most challenge trials with PRRSV and PCV2 perform successive inoculations, several days apart, concomitant infection by both viruses has been shown to act synergistically in SPF piglets, inducing an excessive activation of Toll-like receptors signaling, possibly setting the stage for increased clinical severity of secondary infections 55 . The simultaneous co-infection challenge also matches more closely the field situation, where both viruses may be contemporaneously circulating 22,56 . The PCV2d strain used in the coinfection inoculum is a field strain from 2011 to 2012 outbreaks of PCVADs. In Thailand, the oldest presence of genotype PCV2d has been retrospectively established, and this genogroup became dominant in the Thai swine industry in 2013-2014 47 . Using PCV2d as a challenge strain thus corresponds to the field situation.
In a recent study exploring the protective effect of Ingelvac PRRS MLV in high-health pigs challenged with PRRSV-2 and PCV2, no significant growth differences were observed over the 42 days post-challenge between vaccinated and non-vaccinated groups 57 . No double negative (non-vaccinated and non-challenged) group was included in that study, however. Also, the PRRSV-2 strain used in the present experiment is a highly pathogenic strain 25,48 . In an experiment designed to assess the pathogenicity of a French PRRSV-1 field strain derived from a modified-live vaccine, simultaneous inoculation of PCV2b and either PRRSV-1 strain (either the field or the MLV strain) has been performed in unvaccinated SPF pigs 58 . The group of pigs coinfected with the field PRRSV strain and PCV2 developed more severe lung lesions and produced a higher transmission rate to naïve contacts, than the group coinfected by the MLV and PCV2, but no difference in ADWG was measured between groups, possibly owing to the high health status of the animals and the limited virulence of the PRRSV strain used. In the present results, whether vaccines were injected on 0 DPV or on 0 and 7 DPV, groups that were vaccinated through the ID route presented with a relatively higher or similar ADWG, depending on which IM vaccine they were compared to. This is in line with lung lesion scoring, both at the macroscopic and microscopic scales, where the one group whose score was not statistically different from the NV/NC group were IM-vaccinated (IMTRF0).
No mortality was observed after challenge, as previously observed in MLV-vaccinated pigs challenged with the same HP-PRRSV-2 strain, but without PCV2 48 . Two groups of pigs developed severe hyperthermia after challenge: IMING0/PCVMH7 and IMTRF0, both being groups of pigs vaccinated by the IM route. In the first group, fever occurrence was comparable to that of the NV/CH group in amplitude and duration. In all other MLVvaccinated groups, no significant change in rectal temperature was observed, compared to the NV/NC group. In a recent trial, high-health pigs vaccinated with Ingelvac PRRS MLV have been inoculated with a PRRSV-2 and a PCV2b US strains 4 weeks post-vaccination 57  www.nature.com/scientificreports/ 3 weeks after coinfection (and reduced PRRS clinical signs over the three first week post-challenge). That trial did not include PCV2 vaccinated pigs, and in the present experiments, pigs were sacrificed on 7 DPC, precluding any further observation. Also, the enhanced virulence of a PRRSV MLV-1-like strain by co-infection with PCV2 that has recently been described 58 was not observed in the present experiment. The fact that conventional piglets (rather than SPF) were used might provide an explanation for this difference.
In the non-vaccinated group, challenge induced a PRRSV viraemia peak at 7 days PI, while NV/NC pigs remained virus-free. Two groups (IMING0/PCVMH7 and IMTRF0) developed a viraemia of a comparable amplitude and duration to that of the NV/CH group. However, these groups presented with significantly different histopathological and IHC lung scores. In the IMING0/PCVMH7 group, both scores were significantly lower than those measured in the NV/CH group. This might point to a control of the challenge virus in the lungs-and hence to a more limited inflammatory reaction induced by the HP-PRRSV-2 strain 48,59 than in the NV/CH group, in relation to previous immunization. On the other hand, in the IMTRF0 group, histopathological and IHC lung scores were not significantly different than those in the NV/CH group, which points of a lack of protection towards the heterologous HP-PRRSV-2 challenge strain. Whether or not these observations are associated with the fact that both these groups were those with the highest post-vaccination viraemia is not clear and might deserve further attention. All other vaccination groups had a comparable PRRSV viraemia profile post-challenge, with a transiently increased viraemia, limited to the 5-7 DPC-which points to a successful control of the challenge strain.
A strong PCV2 viraemia was observed in the NV/CH group, while none of the NV/NC animals had detectable PCV2 DNA in their serum, pointing to the absence of accidental natural infection during the trial. None of the vaccinated groups presented a comparable viraemia peak; the IMING0/PCVMH7 and IMTRF0 groups were those with the highest viremic peak among vaccinated groups. Albeit significantly lower than that observed in control animals, it was still significantly higher than that of all other groups (which had a common viraemia pattern). In both IMING0/PCVMH7 and IMTRF0 groups, the IHC lung lesion scores were significantly lower than those of the NV/CH pigs which confirms that the corresponding vaccines provide significant protection against PCV2. Since all commercial PCV2 vaccines included in this trial were designed using a PCV2a stain, the fact that PCV2d might partially escape cross-protection from PCV2a-based vaccines 14 is not relevant to the present study. More probably, co-infection might have triggered PCV2 replication in some pigs or pig groups, as has been previously observed by others 57,60 .
Several vaccination route specific differences were observed. These were in the induction of the IL-10 secretion and IFN-γ-SC induction. ID-vaccinated pigs had significantly lower IL-10 levels and higher IFN-γ-SC frequencies compared to that of the IM-vaccinated pigs regardless of the vaccine use and timing of administration. Regarding cell-mediated immune response (Th1), both the IM and the ID routes are acknowledged to induce specific IFN-γ-SC following MLV injection, which helps reduce PRRSV viraemia 61 . In the present trials, when stimulated with the homologous vaccine strains, IFN-γ-SC were detectable in the serum of all vaccinated groups as early as 3 weeks post-immunization. This was also true for IFN-γ-SC induced by heterogenous (HP-PRRSV-2) stimulation, which agrees with previous experiments 48,54 . Once pigs were challenged with the PRRSV-2 strain, a comparable pattern of continued induction of "homologous" cells was observed in all vaccinated groups. However, the amplitude of the response was significantly stronger in groups that had received the MLV vaccine by the ID route (i.e., IDPP0/PCVMH7 and IDPP0/PCVMH0), which is in line with the recent description of the positive impact of ID over IM routes 62 . This difference disappeared for "heterologous" cells, which were highly induced after challenge in all groups, with the notable exception of the IMTRF0 group. Comparable to others, although with different immunization products, both injection routes provided good protection against the heterologous challenge, while they produced different levels of IFN-γ-SC 62 . Induction of IFN-γ-SC was also observed in the 2 weeks following PCV2 vaccination, upon in vitro stimulation of PBMCs with the heterologous PCV2d strain (all vaccines are based on a PCV2a capsid structure). The PCV2d challenge allowed the induction of such cells to peak at 7 DPC, with a remarkable increase in the IDPP0/PCVMH7 group, well-above the peak obtained in the other groups, and a surprising absence of peak in the IMTRF0 group. In the latter group, this relates to the limited control of PCV2 peak viraemia after challenge. The absence of response in the NV/CH group may by contrast suggest that vaccination in the face of maternal antibodies in the study did induce IFN-γ-SC.
Although all vaccines tested in the present experiments induced a comparable pattern of increase in IL-10 concentration upon PBMC stimulation with homologous strains, there was a difference in the peak of these responses between vaccines, a difference that had previously been highlighted by others in previous studies 63 . In the present results, there seems to be a delay in the IL-10 peak, occurring at 7 DPV in the groups vaccinated by the IM route and at 14 DPV in those vaccinated by the ID route. In comparable immunization protocols (although with different PRRS MLVs), the IL-10 peak was observed at 14 DPV 25 and 21 DPV 48 for both injection routes. It is possible that each MLV strain or even each vaccine formulation has different abilities in triggering effectors of IL-10 secretion. One interesting point is that the IMTRF0 is the group with the highest IL-10 peak. IL-10 being an immunosuppressive cytokine, it suggests that this vaccine might induce a higher IL-10 response and possibly induce an earlier or stronger Treg activation (not measured), explaining the lower levels in the other markers of the adaptive immune response measures in the present experiments. In any case, it illustrates that the context of presentation of the MLV may be modulated by adjuvants, since the vaccine used in the IMTRF0 group combines in a single product for single administration a PRRSV live strain and two inactivated valences (PCV2 and M. hyopneumoniae). Overall, these experiments demonstrate that commercially available multivalent vaccines, with single or two-injection priming protocols, induce protection upon concomitant challenge with HP-PRRSV-2 and PCV2. The ID immunization route provides an improved specific immune response, both through Th1-and Th2-effectors, over the IM route. The limited ability of the MLV used in the IMTRF0 group to mount immunity markers and lesion scores at comparable levels to the other PRRSV MLVs might deserve further investigation, through the exploration of Treg activation pathway. www.nature.com/scientificreports/ Our findings demonstrate that when comparing similar vaccines administered through similar routes with the only difference concurrently or separately, concurrent vaccination of PRRSV and PCV2 antigens was welltolerated without adverse reactions. Differences in the immune responses were observed mainly due to the vaccination route. The ID vaccination had significantly lower IL-10 levels and higher IFN-γ-SC values when compared to the IM-vaccinated groups. In terms of clinical outcomes, only one group (IMPP0/PCVMH7) showed statistically better efficacy compared to its concurrent counterpart, as demonstrated by lower viremia levels, lung lesions, and antigen scores. However, these consequences only appeared in the IM route. When compared to the ID routes, these outcomes disappeared.
In terms of clinical relevance, our study confirms that concurrent vaccination of PRRSV-2 MLV and PCV2 via either the IM or the ID routes could be applicable to assist with the control of PRDC without adding additional reactogenicity. Therefore, producers can select concurrent or separate vaccination depending on the situation on the farm. In vertically integrated operations with multi-site production where lesser health challenges exist, the consumer has higher visibility of production, and meat quality and animal welfare improvements are most important. Concurrent vaccination of PRRSV and PCV2 vaccines via the ID route might be a useful alternative tool for producers wishing to reduce the handling of pigs and hence acute stress while simultaneously controlling PRRSV and PCV2 infections. In this circumstance, intradermal vaccination has several additional benefits, ranging from improved food safety, due to the elimination of needles, to potential biosecurity improvements from the reduction in iatrogenic disease transmission 43 . In situations where the ID vaccination is not possible due to a lack of equipment, the IM vaccination may be the only option available. Based on our findings, if applying IM vaccination, better efficacy results are obtained by splitting up the antigens compared with concurrent vaccination.
Our study contained several limitations. Firstly, it was performed in conventional pigs, owing to the objectives of providing results useful to practitioners and farmers, especially in controlling PRRSV. This might have hampered a clear-cut picture of the adaptive immune response of piglets with MDA against PCV2, but again, this reflects field conditions. Secondly, our common inoculation of both an HP-PRRSV-2 strain and PCV2 might have been seen as an excessive challenge to swine health, but it was approved by the university's ethical committee and reflects a common situation in the field. Also, the fact that no mortality was observed, including in the NV/NC group, which supports our choice. We did not include a Mycoplasma hyopneumoniae challenge in this study because owing to epidemiological reasons, exposure to this pathogen usually occurs later in production 28 . Future studies will add this challenge while maintaining the pre-existing challenge model. Thirdly, it might appear that the delay between challenge and termination of the study (7 days) is too short to see a clinical outcome of the PCV2 challenge. This limitation is substantiated by the observation of PCVAD signs in pigs at 4 weeks postchallenge in another experimental double challenge 57 and by the high IHC PCV2 score observed in the NV/CH group. However, our PCV2 DNA in the blood samples out of 28 DPC serves as an appropriate proxy.

Conclusions
Based on the immune parameters assessed in the study, concurrent vaccination of both PCV2 and PRRSV antigens does not reduce vaccine efficacy or introduce additional reactogenicity. Pigs vaccinated by the concurrent ID route benefitted from equivalent or better immune responses and protection against the dual challenge than those vaccinated by the corresponding separate route. Meanwhile, the protection in the IM route appeared to be better in the corresponding to the separate route than in the concurrent route. The single group that received a trivalent mixed vaccine mounted a more limited response in immunity development and efficacy markers compared with all the other groups.

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.